1. Field of the Invention
The present invention relates generally to the field of cardiovascular medicine. More specifically, the present invention relates to synthetic peptides that can rapidly lower plasma cholesterol through enhanced LDL and VLDL uptake and degradation by cells.
2. Description of the Related Art
Apolipoprotein E (apo E) plays an important role in the metabolism of triglyceride-rich lipoproteins, such as very low density lipoprotein (VLDL) and chylomicrons. Apolipoprotein E mediates the high affinity binding of apo E-containing lipoproteins to the low density lipoprotein (LDL) receptor (apo B, E receptor) and the members of its gene family, including LDL receptor related protein (LRP), very low density lipoprotein receptor (VLDLR) and the apoE2 receptor (apoE2R) (1). The putative and complex role of apo E in atherosclerosis has been emphasized by several observations: (i) mice that overexpress human apo E have lower levels of total plasma cholesterol levels (2), (ii) intravenous injection of human apo E into cholesterol-fed rabbits protects these animals from atherosclerosis (3), and (iii) loss of the apo E gene in mice produces spontaneous atherosclerosis (4) which is ameliorated when macrophage-specific apo E expression is reconstituted in apo E-deficient mice (5).
Apo E is secreted as a 299 amino acid residue protein with a molecular weight of 34,200. Based on thrombin cleavage of apo E into two fragments, a two domain hypothesis was initially suggested to explain the fact that the C-terminal region of apo E (192-299) is essential for its binding to hypertriglyceridemic VLDL and the N-terminal 22 kDa domain (1-191), binds to the LDLR (6). Additional physical-chemical characterization of the protein and its mutants have extended this concept and have shown that the region 192-211 binds to phospholipid while the amino terminal domain (1-191) is a globular structure that contains the LDL receptor binding domain in the H$ (130-166) helix (7). Studies with synthetic peptides (Sparrow et al.) and monoclonal antibodies pinpointed the LDL receptor binding domain of apo E between residues 129-169, a domain enriched in positively charged amino acids, Arg and Lys (8-11).
Further studies with synthetic peptides were used to characterize the structural features of the binding domain of apo E that mediates its interaction with the LDL receptor (10-12). Residues 141-155 of apo E, although containing the positively charged residues, did not compete for binding of LDL in a human skin fibroblast assay, but did so only as tandem covalent repeats [i.e. (141-155)2]. N-acetylation of the [141-15512]2 peptide, on the other hand, enhanced LDL binding to fibroblasts (13). The N-acetylated [141-155]2 analog selectively associated with cholesterol-rich lipoproteins and mediated their acute clearance in vivo (13). Furthermore, these studies indicated that the prerequisite for receptor binding is that the peptides be helical (12). Enhanced LDL uptake and degradation were also observed (14) using synthetic peptides modified to increase lipid association by N,N-distearyl derivation of glycine at the N-terminus of the native 129-169 sequence of Apo E (14). Although LDL binding is mediated by the cationic sequence 141-155 of human Apo E, Braddock et al. (15) have shown that model peptides of the highly conserved anionic domain (41-60 of human Apo E) also modulate the binding and internalization of LDL to cell surface receptors. However, these peptides do not enhance LDL degradation.
Each of the peptides described above used some form of the natural apo E receptor binding sequence. The prior art is deficient in the lack of synthetic peptides that enhance LDL and VLDL uptake. The present invention fulfills this long-standing need and desire in the art.
All of the peptides synthesized in the prior art studies mentioned above made use of the natural apo E sequence. With a view to designing a peptide with minimal structural features of apo E for receptor binding, two essential properties of apo E were incorporated: 1) a lipid binding domain at the C-terminus of the designed peptide, and 2) the receptor binding domain 141-150 from the human apo E sequence at the N-terminus. It was hypothesized that since lipid binding is essential for surface localization of the peptide on lipoproteins and for the receptor binding domain of apo E to be appropriately accessible to bind to the LDL receptor, joining a well-characterized, lipid-associating peptide such as the model class A amphipathic helix, 18A (16), to the 141-150 peptide sequence of apo E should be sufficient to confer biological activity.
A peptide LRKLRKRLLR-18A (hE-18A) is designed, in which LRKLRKRLLR (SEQ ID No. 1) is the 141-150 region of human apo E and 18A is a class A amphipathic helical peptide that associates with phospholipids and lipoprotein surfaces (17). To characterize the role of individual amino acid residues in the peptides several additional analogs were prepared. The receptor binding domain of apo E, LRKLRKRLLR, is well conserved in several species (Table 1). A peptide with the mouse apo E sequence, LRKMRKRLMR-18A (mE-18A, SEQ ID No. 2), where two conserved Leu in hE-18A were changed to Met, was also synthesized. To determine whether the receptor binding is sequence or charge specific, an analog, LRRLRRRLLR-18A (hE(R)-18A, SEQ ID No. 3) was synthesized. The sequences of these peptides are shown in Table 2.
In addition, two-domain peptides were synthesized which were N-terminally protected since Ac-18A-NH2 was previously shown to be more helical with a higher lipid affinity for phospholipids than the free peptide (16). The properties of these N-terminally protected peptides were compared with Ac-18A-NH2, a peptide studied previously (17, 18), as a control peptide. The present invention describes their lipid-associating properties and the effect of these peptides on LDL and VLDL binding to and degradation in HepG2 cells.
Thus, the present invention is directed to the physical-chemical properties and the effects of these peptides on human LDL and VLDL binding and degradation. The studies demonstrate that these dual-domain peptides have unusual ability to bypass the LDLR pathway and the LRP receptor pathway and enhance dramatically the rapid uptake of LDL/VLDL by a cellular pathway that involved heparan sulfate proteoglycan. This peptide-enhanced LDL/VLDL uptake pathway suggests an alternate pharmacological route for LDL/VLDL cholesterol lowering independent of expression of LDLR family members.
In one embodiment of the present invention, there is provided a synthetic apolipoprotein E-mimicking peptide comprising a receptor binding domain of apolipoprotein E covalently linked to a lipid-associating peptide. Preferably, the lipid-associating peptide is model class A amphipathic helical peptide 18A. Further, it is preferable that the synthetic peptide is N-terminally protected using acetyl and amino groups.
In another embodiment of the present invention, the receptor binding domain of apolipoprotein E has an amino acid sequence selected from the group consisting of SEQ ID Nos. 1-3 and apolipoprotein E is from species selected from the group consisting of human, mouse, rabbit, monkey, rat, bovine, pig and dog.
In still another embodiment of the present invention, the synthetic apolipoprotein E-mimicking peptide both enhances LDL/VLDL binding to cells and increases LDL/VLDL degradation by cells, e.g., by cells such as hepatocytes.
In still another embodiment of the present invention, there is provided a method of using the synthetic apolipoprotein E-mimicking peptide to rapidly reduce circulating plasma cholesterol in vivo.
In yet another embodiment of the present invention, there is provided a pharmaceutical composition comprising a synthetic apolipoprotein E-mimicking peptide and a pharmaceutically acceptable carrier.
The present invention is further directed to applications of the pharmaceutical composition disclosed herein in enhancing LDL/VLVL binding to a cell, increasing LDL/VLDL degradation by a cell, lowering LDL/VLDL cholesterol in an in-need individual o r treating an individual with atherosclerosis by administering to the cell or individual with a pharmacologically effective dose of the pharmaceutical composition. Preferably, the effective dose is from about 0.01 mg/kg to about 100 mg/kg.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.